This research focuses on understanding the basis for the biochemical abnormalities in the porphyrias, and defining the pathogenesis of liver disease in some of these disorders. Three areas will be examined: (1) Particular attention is paid to protoporphyria, a disorder in which there is a defect in ferrochelatase, the terminal enzyme of the heme biosynthesis pathway. The disease is inherited in humans as an autosomal dominant trait. The disease has also been found in cattle, where it is inherited in a homozygous fashion. A kinetic ELISA assay has been developed which will be used to quantify the amount of ferrochelatase protein in human and bovine tissues. The possibility exists that protoporphyria in humans results from more than one type of mutation which may be differentiated by the amount of immunoreactive protein present. Peptide analysis of ferrochelatase will be performed in order to compare the normal and protoporphyria enzymes. Initial studies will be done with the bovine enzymes, since this is facilitated by the homozygous defect in ferrochelatase in bovine protoporphyria. Emphasis will be on identifying the peptide sequences which contain the active site of the enzyme. Molecular studies of the ferrochelatase defect will also be initiated. Bovine cDNA libraries obtained from both normal and protoporphyria tissues will be screened, and a clone containing the cDNA coding for ferrochelatase will be isolated The cDNA will be sequenced in order to infer the complete peptide sequences for normal and protoporphyria enzymes. Comparison of the primary sequences will determine whether bovine protoporphyria is due to a point mutation in the structural gene for ferrochelatase. The cDNA for normal bovine ferrochelatase will also be used to probe for human ferrochelatase cDNA and initiate studies of the molecular defect in the human condition. (2) The activity of ferrochelatase in the mitochondrial is likely to be influenced by its lipid environment. This will be examined using cultured cells which are incubated with media containing specific fatty acids. The lipid effect will also be examined by incorporating purified ferrochelatase into liposomes of defined composition. The results may suggest a dietary means of modulating ferrochelatase activity in vivo (3) A mechanism for hepatocellular damage in porphyria cutanea tarda is postulated in which the oxidation of uroporphyrinogen to uroporphyrin in the presence of molecular oxygen and iron causes the formation of activated oxygen species which damage cellular membranes. This will be tested using a model system composed of liposomes, uroporphyrinogen, and various metals. The interaction of antioxidants with this system will also be studied. The results of this of this study may provide a rationale for using antioxidant therapy in porphyria cutanea tarda in humans.